PROJECT SUMMARY Diseases of the biliary system are uncommon, however, collectively they comprise a significant percentage of the indications for liver transplantation in adult and pediatric populations. The pathogenesis of these disorders likely involves cellular responses to the harmful effects of bile, to which epithelial cells lining the bile ducts are continuously exposed. Bile toxicity arises largely from the oxidative damage caused by bile salts, whose detergent properties disrupt intracellular organelles, particularly mitochondria. Bile duct epithelial cells, also known as cholangiocytes, have evolved strategies for preventing bile toxicity in addition to having robust anti-oxidant defense systems, which are present in all cells to combat reactive oxygen species generated during normal metabolism. Recent work from my laboratory using the zebrafish system has identified regional susceptibilities to redox stress imparted by the biliary toxin biliatresone, which is an isoflavone recovered from Dysphania species plants responsible for outbreaks of epidemic biliary atresia (BA) in livestock. We have identified differences in the glutathione redox state in extra-hepatic and intra-hepatic cholangiocytes (EHC; IHC), and their ability to mitigate oxidative stress. This suggests that variation in cholangiocyte redox defenses may play a role in the pathogenesis of other biliary diseases. The goal of this proposal is to study the mechanisms responsible for this variation by exploiting the strengths of the zebrafish system. In aim 1, we will examine basal and stress-induced changes in glutathione metabolism in IHC and EHC. In aim 2, we will examine transcriptional mediators of basal and inducible stress responses in IHC and EHC, and determine whether they evolve from developmentally encoded differences in epigenetic regulators of gene transcription. In aim 3, we will examine the role of proteomic stress responses in redox induced biliary injury, focusing on a co- chaperone we have linked to human BA. In aim 4, we will study the differential effects in IHC and EHC of IDH gene mutations found in human bile duct cancers that have been shown to alter NADPH and glutathione metabolism in other cancers. The long term goal of these studies is to translate discoveries made in the zebrafish to humans using mammalian in vivo and cell culture models.